Insulin is thought to elicit its effects by crosslinking the two extracellular ␣-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.I nsulin is one of the most studied peptide hormones because of its importance in maintaining glucose homeostasis. This 51-aa hormone is very well characterized with regard to its structure, both in crystal form and in solution. The insulin receptor (IR) is a transmembrane ␣ 2  2 glycoprotein whose intracellular tyrosine kinase domain is activated by binding of insulin, leading to a cascade of intracellular signaling events. The kinase domain of the IR (1) and an extracellular fragment of the related receptor for insulin-like growth factor I (IGF-IR; ref. 2) have been crystallized, but the structure of the insulin binding domain of the IR is not known, and the mechanism for the transmission of a signal through its transmembrane domain is not well understood. A model for the binding and activation has been proposed in which insulin uses two different sites on its surface to crosslink the two ␣-subunits of the IR, thus inducing a conformational change that activates the receptor (refs. 3 and 4; Fig. 1).In a previous report (5), we panned random, highly diverse peptide display libraries against the IR. By using this approach, we identified a large number of peptides binding to the IR and competing for insulin binding with micromolar or submicromolar affinity, although these peptides had no sequence homology with insulin. These peptides bound to two discrete hotspots on the receptor (designated site 1 and site 2), and these hotspots appeared to correspond to the two contact sites involved in insulin binding predicted by the crosslinking model (ref. 3 and J.B., unpublished results). At least two different sequence motifs were found for site 1 peptides, and some of these were full agonists but of low affinity. Other site 1 peptides were antagonists, whereas site 2 peptides were either antagonists or inactive. The mechanism behind the agonism of the site 1 peptides is not known, but it has been speculated that site 1 binding may be important for receptor activation, whereas the role of the site 2 interaction may be more related to affinity and selectivity. In addition to these two families of peptides, a third group was identified, but no further work has been done on this group. In the present work, we have used site 1 and site 2 peptides as building blocks ...
We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor. All of the peptides competed for insulin binding and had affinity constants in the high nanomolar to low micromolar range. Based on competition studies, peptides were grouped into non-overlapping Sites 1, 2, or 3. Some Site 1 peptides were able to activate the tyrosine kinase activity of the insulin receptor and act as agonists in the insulin-dependent fat cell assay, suggesting that Site 1 marks the hotspot involved in insulin-induced activation of the insulin receptor. On the other hand, Site 2 and 3 peptides were found to act as antagonists in the phosphorylation and fat cell assays. These data show that a peptide display can be used to define the molecular architecture of a receptor and to identify the critical regions required for biological activity in a site-directed manner.
Double stranded DNA sequencing of plasmids isolated from minipreps is now a routine practice. Here I provide a procedure for sequencing double stranded DNA using Sequenase (USB) where the sample maneuvering time can be reduced tremendously.Conventionally, the DNA isolated from miniprep cultures is treated to remove RNA, followed by alkaline denaturation, neutralization and precipitation steps before being dissolved and hybridized to primers. The routine is rather time consuming especially when a large number of samples is involved.The protocol described here modifies the steps after pelleting the miniprep DNA. It combines alkaline denaturation of the DNA, alkaline hydrolysis of RNA and annealing of the template and primers into a single step; thus RNAse digestion and phenol/chloroform extraction are totally eliminated. Furthermore, it does not require the DNA to be precipitated after alkaline denaturation. The result of these modifications not only saves time and improves the yield, but also reduces the exposure to hazardous chemicals.The quality of the sequence information produced by this method is excellent (Figure 1). This method is presented in a step by step protocol as follows:1. Dissolve the miniprep DNA pellet in 25 to 50 A1 of TE buffer depending on the DNA yield (In my hands, the amount of DNA yield from the rapid-boiled method (1) is about twice as much as that of the alkaline-lysis method (2), i.e., 50 1l for the rapidboiled method and 25 1l for the alkaline-lysis method). Use 5 1l per sequencing reaction. 2. Add 1 yd of sequencing primers (10 ng/lI).3. Add 1 Al of 1N NaOH and mix by pipetting.
The polymerase chain reaction (PCR) is a powerful technique for amplifying specific DNA sequences that has become a routine procedure in many laboratories. Many strategies for subcloning PCR products have been developed, but most require the use of restriction enzymes and/or kinase treatment prior to ligation. These 'conventional' subcloning strategies may be time consuming and inefficient. A recently described method (1) which
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